Diversity structure of the microbial communities in the guts of four neotropical termite species.

The termite gut microbiome is dominated by lignocellulose degrading microorganisms. This study describes the intestinal microbiota of four Argentinian higher termite species with different feeding habits: Microcerotermes strunckii (hardwood), Nasutitermes corniger (softwood), Termes riograndensis (soil organic matter/grass) and Cornitermes cumulans (grass) by deep sequencing of amplified 16S rRNA and ITS genes. In addition, we have performed a taxonomic and gut community structure comparison incorporating into the analysis the previously reported microbiomes of additional termite species with varied diets. The bacterial phylum Spirochaetes was dominant in the guts of M. strunckii, N. corniger and C. cumulans, whereas Firmicutes predominated in the T. riograndensis gut microbiome. A single bacterial genus, Treponema (Spirochaetes), was dominant in all termite species, except for T. riograndensis. Both in our own sequenced samples and in the broader comparison, prokaryotic α-diversity was higher in the soil/grass feeders than in the wood feeders. Meanwhile, the ß-diversity of prokaryotes and fungi was highly dissimilar among strict wood-feeders, whereas that of soil- and grass-feeders grouped more closely. Ascomycota and Basidiomycota were the only fungal phyla that could be identified in all gut samples, because of the lack of reference sequences in public databases. In summary, higher microbial diversity was recorded in termites with more versatile feeding sources, providing further evidence that diet, along with other factors (e.g., host taxonomy), influences the microbial community assembly in the termite gut.

Comparative Pathogenesis of Generalist AcMNPV and Specific RanuNPV in Larvae of Rachiplusia nu (Lepidoptera: Noctuidae) Following Single and Mixed Inoculations.

The South American soybean pest, Rachiplusia nu (Guenée), is naturally infected by Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and Rachiplusia nu nucleopolyhedrovirus (RanuNPV). We compared their pathogenicity to fourth-instar R. nu larvae, by evaluating time to death and virus spread throughout the tissues in single and mixed infections. Bioassays showed that generalist AcMNPV had a faster speed of kill than specific RanuNPV, while the mixed-virus treatment did not statistically differ from AcMNPV alone. Histopathology evidenced similar tissue tropism for both viruses, but co-inoculation resulted in mostly AcMNPV-infected cells. In sequential inoculations, however, the first virus administered predominated over the second one. Implications on baculovirus interactions and biocontrol potential are discussed.

Potential of predatory Neotropical ladybirds and minute pirate bug on strawberry aphid.

Laboratory trials were performed to determine the impact of three Neotropical predatory coccinellids (Cycloneda sanguinea, Eriopis connexa and Coleomegilla quadrifasciata) and a minute pirate bug (Orius insidiosus) on Chaetosiphon fragaefolii, an important strawberry aphid pest. The predation on C. fragaefolii nymphs and adults, as well as the time to the first attack of all predators were compared with predation on Aphis gossypii. Predator preferences for prey and aphid defensive behavior were also evaluated. Moreover, the effect of coccinellids on C. fragaefolii population growth was assessed in experimental greenhouse conditions. The predation rate varied among predators, being significantly lower for O. insidiosus than for the coccinellids. Consumption was higher on A. gossypii than on C. fragaefolii, regardless of the aphids developmental stage. The time to the first attack of all predators was longer in the presence of C. fragaefolii. Walking away and cornicle secretion were the most common antipredator behaviors of aphid against coccinellids and O. insidiosus, respectively. Coccinellids preferred A. gossypii over C. fragaefolii, while O. insidiosus showed indifference. Cycloneda sanguinea and E. connexa exhibited the highest suppression effect on the growth rate of C. fragaefolii. Thus, the four predators evaluated could contribute to reduce strawberry aphid populations, especially C. sanguinea and E. connexa.

Biological and molecular characterization of Rachiplusia nu single nucleopolyhedrovirus, a promising biocontrol agent against the South American soybean pest Rachiplusia nu.

Rachiplusia nu (Lepidoptera: Noctuidae) is a key soybean pest in Argentina. Current management of this moth relies mainly on the use of synthetic insecticides and transgenic plants. In search of biological control-based alternatives, a baculovirus from R. nu (hereafter RanuNPV) was characterized and its insecticidal properties tested under laboratory conditions. RanuNPV occlusion bodies (OBs) were nearly tetrahedral, averaging 1.0 ±â€¯0.2 µm in their longest edge and containing singly enveloped nucleocapsids. Histopathology of infected late-instar larvae revealed broad tissue tropism, where fat bodies and epidermis were the most affected organs. Phylogenetic analysis of concatenated polh, lef-8 and lef-9 partial sequences classified RanuNPV as a new species that clusters with other group II alphabaculoviruses infecting larvae of Plusiinae. Bioassays performed with R. nu neonates determined the median lethal dosage to be approximately 2.5 OBs/larva; most insects died within 4-5 days post inoculation showing typical baculovirus-induced liquefaction. No effects were observed in other lepidopteran species assayed, including Spodoptera frugiperda, Cydia pomonella and Diatraea saccharalis. High pathogenicity and host specificity make RanuNPV a good candidate for controlling R. nu.

The autographa californica multiple nucleopolyhedrovirus Ac12: A non-essential F box-like protein that interacts with cellular SKP1 component of the E3 ubiquitin ligase complex.

The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac12 gene, which is conserved in ten other baculovirus, codes a predicted 217 amino acid protein of unknown function. In this study, we investigated the role of ac12 during baculovirus infection, by generating an ac12 knockout virus. The transfection of the recombinant genome in insect cells resulted in unaltered viral dispersion and occlusion body production when compared to the control bacmid. This finding demonstrates that ac12 is a non-essential gene. Transmission and scanning electron microscopy (SEM) analyses showed that ac12 knockout virus produced occlusion bodies morphologically similar to those obtained with the control and capable to occlude virions. However, a slight but significant size difference was detected by SEM observation of purified occlusion bodies. This difference suggests that ac12 may be involved in regulatory pathways of polyhedrin production or occlusion body assembly without affecting either viral occlusion or oral infectivity in Rachiplusia nu larvae. This was evidenced by bioassays that showed no significant differences in the conditions tested. A qPCR analysis of viral gene expression during infection evidenced regulatory effects of ac12 over some representative genes of different stages of the viral cycle. In this study, we also showed that ac12 is transcribed at early times after infection and remains detectable up to 72 hours post-infection. The mRNA is translated during the infection and results in a protein that encodes an F-box domain that interacts in vivo and in vitro with S phase kinase associated protein 1 (SKP1) adaptor protein, which is potentially involved in protein ubiquitination pathways.

Biological and molecular features of Nosema rachiplusiae sp. n., a microsporidium isolated from the neotropical moth Rachiplusia nu (Guenée) (Lepidoptera: Noctuidae).

Light, electron microscopy and DNA analyses were performed to characterize a microsporidium infecting Rachiplusia nu larvae from a laboratory rearing in Argentina. Diplokaryotic spores were oval and measured 3.61 ± 0.29 × 1.61 ± 0.14 µM (fresh). The spore wall was composed of an electron-dense exospore and an electron-lucent endospore, ca. 30 nm and 100-120 nm thick, respectively. The polar filament was arranged in a single rank of 10-12 coils (typically 11). Microsporidian cells were found in the cytoplasm, next to the endoplasmic reticulum (especially the prespore stages) and generally surrounded by electron-lucent spaces. The infection was polyorganotropic; the fat body appeared as the most heavily invaded tissue, followed by tracheal matrix and epidermis. A molecular phylogeny based on the small (SSU) and large subunit (LSU) ribosomal RNA genes clearly placed the new isolate within the "Nosema bombycis clade". Considering both SSU and LSU concatenated partial sequences, the microsporidium from R. nu showed 99.5% nucleotide similarity with N. bombycis and 99.8% with its closest relative, a microsporidium isolated from Philosamia cynthia. According to its genetic and biological features, the R. nu isolate is proposed as the new species Nosema rachiplusiae sp. n., expanding the limited knowledge on microsporidia associated to endemic South-American moths.

Further Description of Helicoverpa zea (Lepidoptera: Noctuidae) Male Genitalia and New Genetic Evidence of Synonymy With Respect to the Anomalous Form, "Heliothis stombleri".

The Helicoverpa/Heliothis complex can cause serious damage to agricultural crops. Phenotypic similarity makes it difficult to discriminate between closely related Helicoverpa species. Currently, morphology of the male genitalia complemented with molecular techniques constitutes the best approach for species identification. In this work, a broad microscopic examination of adult Helicoverpa zea (Boddie) males (n = 200) captured in central Argentina was carried out in order to provide a detailed description of the valvae and the phallus. A considerable degree of variability was recorded. Both rounded and sharp valve apices were observed and valvae were not always parallel-sided. Most evident differences were detected concerning the number of cornuti on the phallus. A range of 15-21 cornuti per phallus was recorded, the mode being 18. A significant minority of the samples (3.5%) displayed an abnormal genital condition showing a constricted phallus lacking cornuti, and pointed valvae. This form was initially attributed to a distinct species, Heliothis stombleri, and later proposed as a synonym of H. zea based on additional morphological observations and molecular studies. Here, a phylogenetic analysis combining mitochondrial (cytochrome oxidase subunit I) and nuclear (elongation factor -1 alpha) genes was performed on these and other Helicoverpa specimens collected in the same geographical region, in order to further verify the taxonomic status of H. stombleri. The tree topology clearly grouped H. stombleri with H. zea, supporting the assumption that the former represents, in fact, an anomalous form of the latter. Further experiments are needed to clarify the etiology of this anomaly and its persistence over time.

Molecular Identification of Helicoverpa armigera (Lepidoptera: Noctuidae: Heliothinae) in Argentina and Development of a Novel PCR-RFLP Method for its Rapid Differentiation From H. zea and H. gelotopoeon.

Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae: Heliothinae) is among the most voracious global pests of agriculture. Adults of this species were identified recently in northern Argentina by dissection of male genitalia. In this work, a rapid and simple molecular tool was designed to distinguish H. armigera from the morphologically similar indigenous bollworms Helicoverpa zea (Boddie) and Helicoverpa gelotopoeon (Dyar), regardless of the life stage. Amplification of partial COI gene with a new primer pair, and subsequent digestion with endonuclease HinfI, yielded different RFLP profiles for the three main Helicoverpa pests currently present in South America. The method was validated in Helicoverpa specimens collected across Argentina, whose identity was further corroborated by COI sequencing and phylogenetic analysis. The data reported here constitute the first molecular confirmation of this pest in the country. The survey revealed the occurrence of H. armigera in northern and central Argentina, including the main soybean- and maize-producing area.

Molecular identification of the secondary endosymbiont Hamiltonella defensa in the rose-grain aphid Metopolophium dirhodum.

This is the first report of the association between the rose-grain aphid Metopolophium dirhodum, a potentially important cereal pest and the facultative symbiont Hamiltonella defensa. The infection with this gamma-proteobacterium was determined by PCR in laboratory-reared and field-collected specimens of an Argentinian population of the aphid. Partial bacterial 16S, IGS and 23S rRNA genes were sequenced and compared to other available Hamiltonella sequences by phylogenetic analysis. the present study provides new information on previously unknown M. dirhodum microbiota.

Immunodetection and subcellular localization of Mal de Río Cuarto virus P9-1 protein in infected plant and insect host cells.

Mal de Río Cuarto virus (MRCV), a member of the genus Fijivirus, family Reoviridae, has a genome consisting of 10 dsRNA segments. The segment 9 (S9) possesses two non-overlapping open reading frames (ORF-1 and ORF-2) encoding two putative proteins, MRCV P9-1 and MRCV P9-2, both of unknown function. The MRCV S9 ORF-1 was RT-PCR amplified, expressed in pET-15b vector, and the recombinant protein produced was used to raise an antiserum in rabbit. Western blot with the specific MRCV P9-1 antiserum detected a protein of about 39 kDa molecular weight present in crude protein extracts from infected plants and insects. However, no reaction was observed when this antiserum was tested against purified virus. In contrast, only virus particles were detected by a MRCV-coat antiserum used as a validation control. These results suggest that MRCV S9 ORF-1 encodes a non-structural protein of MRCV. Immunoelectron microscopy assays confirmed these results, and localized the MRCV P9-1 protein exclusively in electron-dense granular viroplasms within the cytoplasm of infected plants and insects cells. As viroplasms are believed to be the replication sites of reoviruses, the intracellular location of MRCV P9-1 protein suggests that it might be involved in the assembly process of MRCV particles.